Microarray techniques for nucleic acid expression analyses

ABSTRACT

Provided are DNA microarray techniques that allow hybridization without RNA amplification, without using cDNA, and without labeling the nucleic acid prior to hybridization. Referred to as the  D ouble-stranded  E xonuclease  P rotection (DEP) assay, the technique permits the sample RNA to be used directly for hybridization, without manipulation in any way. Further provided is a microarray technique for high-throughput miRNA gene expression analyses, termed the  R NA-primed,  A rray-based,  K lenow  E nzyme (RAKE) assay. The RAKE assay is a sensitive and specific technique for assessing single-stranded DNA and RNA targets, and offers specific advantages over Northern blots.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional PatentApplication No. 60/612,301, filed Sep. 22, 2004, the content of which isincorporated herein by reference.

GOVERNMENT INTERESTS

The present invention was supported in part by U.S. National Institutesof Health Training Grant T32-AG00255. The government may have certainrights in the invention.

FIELD OF THE INVENTION

The invention relates to the field of microarray techniques.

BACKGROUND OF THE INVENTION

RNA expression profiling is used to characterize the RNA species presentin a sample. Many different techniques are used for this task, eachhaving its own strengths and weaknesses. DNA microarrays are one of thebest ‘high-throughput’ techniques for RNA expression profiling. However,most DNA microarrays require extensive sample manipulation.Specifically, sample RNA must be reversed-transcribed into cDNA, thenamplified and labeled with one or more fluorophores prior to arrayhybridization. As a result, biases may be introduced in any of thesesteps that may artificially skew the results of the microarray analysis.In addition, the current microarray technology is limited in that it isdesigned to detect only mRNAs.

A major fraction of cellular RNAs, comprise noncoding RNAs, many ofwhich have regulatory functions. Detection of these noncoding RNAsrequire (or would benefit from) development of novel microarraytechnology that would not require sample RNA amplification or labeling.Such technology would be desirable, for example, in the detection ofmicroRNAs (miRNAs).

MicroRNAs are small (˜22 nucleotide) regulatory RNAs, that are found inthe vast majority of eukaryotic cells. MiRNAs play important roles inplant and animal development, apoptosis, fat metabolism, growth controland hematopoietic differentiation (Lee et al., Cell 116 S89-92, 81 pfollowing S96 (2004); Ruvkun et al., Cell 116, S93-96, 92 p followingS96 (2004); Lagos-Quintana et al., Science 294:853-858 (2001); Bartel etal., Cell 116:281-297 (2004); Nelson et al., Trends Biochem. Sci.28:534-540 (2003); Carrington et al., Science 301:336-338 (2003)).Dysregulation of miRNAs may contribute to human disease including cancerCalin et al., Proc. Natl. Acad. Sci. USA 99:15524-15529 (2002); Michaelet al., Mol. Cancer Res. 1:882-891 (2003); Takamizawa et al., CancerRes. 64:3753-3756 (2004)).

Many individual miRNAs are conserved across widely diverse phyla,indicating their physiological importance. More than 200 miRNAs havebeen reported thus far from mammals, and miRNAs are estimated to accountfor ˜1.0% of expressed human genes (Bartel et al., 2004). Most animalmiRNAs have the capacity to regulate multiple mRNA targets (Kiriakidouet al., Genes Dev. 18(10); 1165-1178 (2004); reviewed in Bartel et al.,2004). Yet such RNAs cannot be studied using conventionalmicroarray-based techniques.

In mammalian cells, primary miRNA transcripts (pri-miRNAs) are cleavedsequentially in the cell nucleus and transported to the cytoplasm (aspre-miRNAs) where mature miRNAs are generated (Lee et al. Nature 425:415-419 (2003)). Mature miRNAs guide regulatory proteins to inducetranslational repression or degradation of specific target mRNAs (Bartelet al., 2004; Murchison et al., Curr. Opin. Cell Biol. 16:223-229(2004)).

High-throughput miRNA gene expression analysis has proven to betechnically challenging. The short length and uniqueness of each miRNArender many conventional tools ineffective. Very small RNAs aredifficult to reliably amplify or label without introducing bias (Ohtsukaet al. Eur. J. Biochem. 81:285-291 (1977); Romaniuk et al. Eur. JBiochem. 125:639-643 (1982)). Prior attempts at systematic geneexpression analysis have involved dot-blots (Krichevsky et al., RNA9:1274-1281 (2003)) or Northern blots (e.g., Sempere et al., GenomeBiol. 5:R13 (2004); Lim et al., Genes Dev. 17:991-1008 (2003)).Additional assays for sensitive detection of miRNAs or their precursorshave been developed, involving realtime quantitative PCR-based analysisof pre-miRNA expression (Schmittgen et al., Nucleic Acids Res. 32:e43(2004)), or a modification of the Invader assay for miRNA detection andquantitation (Allawi et al., RNA 10:1153- 1161 (2004).

While Northern blots are currently the gold standard of miRNA validationand quantification (Ambros et al., RNA 9:277-279 (2003)), thespecificity of the Northern blot technique has received scant criticalreview. This is surprising considering the widespread use of the method.Short DNA/RNA hybrids demonstrate T_(m) and binding dynamics that varysignificantly with probe and target nucleotide composition, buffercontents, and the time and temperature of hybridization (Dai et al.,2002; Liu et al, 2001; Dorris et al., 2003; Urakawa et al., Appl.Environ. Microbiol. 69:2848-2856 (2003); Guschin et al., Appl. Environ.Microbiol. 63:2397-2402 (1997)). Thus, it is highly probable that“signal intensity” for Northern blots will vary from one miRNA sample toanother, as well as from one experiment to another. Moreover, standardNorthern blotting does not provide absolute quantification, meaning thateach RNA queried must include a standard curve in order to be considered“absolutely quantitative,” and each standard curve must further be runin parallel with each individual experiment (Lim et al., 2003, supra).

Because higher-throughput techniques involving mature miRNAs are neededto further understand the role(s) played by miRNAs in normal and diseasetissues, two groups have reported work on microarrays for miRNAs. Croceand colleagues reported an oligonucleotide microarray for miRNA andpre-miRNA profiling (Liu et al., Proc. Natl. Acad. Sci. USA 26:9740-9744(2004); Liu et al., Proc. Natl. Acad. Sci. USA 32:11755-11760 (2004)),wherein the assay involves the use of a biotinylated primer containing arandom octamer sequence at the 3′-end. The Liu et al. primer is used,along with reverse transcriptase, to generate a cDNA library from totalRNA. The cDNA is isolated and applied to a microarray containingcovalently linked DNA oligonucleotide probes corresponding to 245 humanand mouse miRNAs (Liu et al., 2004). Horvitz and colleagues alsoprepared cDNAs from miRNAs using techniques previously employed for thecloning of miRNAs. This was accomplished by ligating adapters to miRNAsusing T4 RNA ligase, followed by R.T-PCR using fluorescently-labeledprimers complementary to the adapters (Miska et al., Genome Biol. 5:R68(2004)). However, while these reported microarray techniques allow forsensitive, specific and high-throughput miRNA expression profiling,e.g., Miska et al., reported a sensitivity of 0.1 fmoles), but thetechnique also requires PCR amplification of the miRNA sample.

Nevertheless, much remains unknown about miRNA biology. For example, themiRNA genes expressed in most tissues, species, and cell lines, are notknown, and the physiological functions-and regulation-of almost allmiRNAs remain to be determined. MiRNAs may also play roles in humandisease that have not yet been explored. These and other topics will beeasier to address experimentally when miRNA gene expression studiesbecome more feasible, and a need in the art has remained until thepresent invention for simple and reliable DNA microarray techniques thatallow for hybridization without RNA amplification or degredation,without the cumbersome steps involved in making and using cDNA, andwithout the need to label the nucleic acid prior to hybridization.

SUMMARY OF THE INVENTION

The present invention fulfills this need, among others by providing DNAmicroarray techniques that allow hybridization without RNAamplification, without using cDNA, and without labeling the nucleic acidprior to hybridization. Referred to as the Double-stranded ExonucleaseProtection (DEP) assay, the technique permits the sample RNA to be useddirectly for hybridization, without manipulation in any way. Thiseliminates biases introduced during sample RNA manipulation usingconventional microarray technology, and greatly facilitatesexperimentation, wherein the novel microarray technology may be used forthe detection of any RNA (including mRNAs and other noncoding RNAs).

Further provided, is a second novel microarray technique forhigh-throughput miRNA gene expression analyses, termed the RNA-primed,Array-based, Klenow Enzyme (RAKE) assay. The RAKE assay is a sensitiveand specific technique for assessing single-stranded DNA and RNAtargets, with specific advantages over Northern blots. Moreover, thisassay may be modified to have broader applications, e.g., RNA profiling,including profiling and quantification of viruses. The availability ofthe novel techniques permits, for the first time, a robust sampling ofmiRNAs made from formalin-fixed, paraffin-embedded (FFPE) pathologicalsamples, followed by expression analysis with RAKE.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description, examples and figures whichfollow, all of which are intended to be for illustrative purposes only,and not intended in any way to limit the invention, and in part willbecome apparent to those skilled in the art on examination of thefollowing, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the RNA-primed, Array-based, KlenowEnzyme (RAKE) assay. The sample probe (SEQID No:3) at the top of thefigure illustrates the generic structure of the DNA oligonucleotidesused on the microarray. The nucleotides at the 5′ side comprise aspacer, which is constant for all the probes, followed by threethymidine nucleotides. The variable portion of each probe is at the 3′end, which is the antisense sequence of various miRNAs.

FIGS. 2A-2C show the sensitivity and dynamic range of RAKE, and providea comparison to matching Northern blots. FIG. 2A shows that indicatedmolecules of a synthetic miRNA (miR-124a, not expressed in HeLa cells)were added to a “complex RNA mixture” derived from low molecular weightHeLa RNAs (10⁸ molecules=0.16 fmoles). A DNA oligonucleotidecorresponding to a plant miRNA (miR-157; not found in HeLa cells) wasalso added at a constant concentration, as a ‘spike-in.’ In the datashown in FIG. 2B, the same number of miR-124a molecules were resolved ona 20% denaturing polyacrylamide gel and analyzed by Northern blots. FIG.2C graphically quantification and comparison of sensitivity between RAKEand Northerns. For RAKE, the signal was defined as the median offoreground spot fluorescence at 532 nm wavelength minus background(defined by surrounding pixel intensity). Negative data values werenormalized to zero, but not otherwise normalized. Each concentrationpoint (solid diamond) represents the mean from 12 spots (two microarrayslides with six spots per miRNA on each microarray. Standard deviationis shown. For Northerns, the signal intensity was measured with aphosphorimager after overnight exposure. Each concentration spot (solidsquare) represents the mean of two different experiments.

FIGS. 3A and 3B are photographic images of representative agarose gelsused to characterize the RNA used in the RAKE assay. “Fr” represents RNAfrom fresh brain tumor, whereas “FFPE” represents RNA fromformalin-fixed, paraffin-embedded brain tumor. FIG. 3A depicts ananalysis of total RNA from fresh and FFPE anaplastic oligodendrogliomatissue on 1% agarose gels. Ribosomal RNAs (28S and 18S) are indicated.FIG. 3B depicts an analysis of low molecular weight RNA from fresh andFFPE anaplastic oligodendroglioma tissue on 3% agarose gels. “T”=totalRNA; “L”=larger RNA only, and “S”=low molecular weight RNA, used for theRAKE assay. The lanes labeled “FFPE” (tumor sample), represent ⅕th ofthe RNA isolated from a single 50 micron-thick section (approximately 1μg of RNA).

FIG. 4 contains a series of representative images from RAKE assays.Samples are indicated on top. Rep 1, Rep 2: replicates. Position andidentity of the spotted probes are shown on the bottom.

FIG. 5 shows the profiling and relative abundance of different miRNAsusing RAKE. H=HeLa; J=Jurkat; M=malignant meningioma; PO=FFPE tissuefrom anaplastic oligodendroglioma; FO=Fresh tissue from anaplasticoligodendroglioma. Light gray squares represent no signal, black squaresrepresent low signal, and cross-hatched squares represent highestsignal.

FIGS. 6A-6C graphically compare results from RAKE assays betweendifferent samples. Numbers on the ordinate and abscissa relate to signalintensities in reference to individual miRNAs (spots). FIG. 6A shows acomparison between different biological replicates in Jurkat cells(R2>0.9). FIG. 6B shows a comparison between Jurkat cells and freshanaplastic oligodendroglioma tissue. Notably, in contrasting FIG. 6Awith 6B, there is poor correlation in the expression levels of miRNAsbetween the two samples, (R2=<0.2), yet many miRNAs are highly expressedin both tissues. FIG. 6C shows a comparison between RAKE signals fromanaplastic oligodendroglioma RNA isolated from FFPE or from freshmaterial (R2>0.9).

FIG. 7 depicts the correlation between Northern blots and RAKE assaysfor HeLa (H), Jurkat (J), malignant meningioma (M), and anaplasticoligodendroglioma (0), 5 μg total RNA loaded per lane. The quality ofthe total RNA is assessed by electrophoresis on 1% Agarose gels,followed by ethidium bromide (EtBr) staining (a representative gel isshown as an insert at the bottom of FIG. 7). Northern blots are shown onthe left; corresponding RAKE assay results are shown on the right.

FIGS. 8A thru 8D demonstrate that RAKE is superior to Northern blots indiscriminating between miRNA paralogs. FIG. 8A provides nucleic acidsequences (SEQID Nos:1, 2 and 4-7) of the three pairs of paralogousmiRNAs tested. 0.1 pmoles (6×10¹⁰ molecules) of each synthetic,paralogous miRNA was analyzed by RAKE or Northern blots. Representativeexperiments for the hsa-miR-23a/23b pair, SEQID Nos:1 and 2,respectively, are shown in FIGS. 8B and 8C for RAKE and Northerns,respectively. In FIG. 8D, the mean signal intensity for each miRNA wasdetermined using RAKE (n=12) and Northern blots (n=2), and a ratiobetween the paralogous miRNAs was calculated for each pair, asindicated.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

The present invention provides DNA microarray techniques that allowhybridization of nucleic acids without RNA amplification, without usingcDNA, and without labeling the nucleic acid prior to hybridization. Theterm “nucleic acid” as used herein, may include any polymer or oligomerof pyrimidine and purine bases, preferably cytosine, thymine, anduracil, and adenine and guanine, respectively. See Lehninger, PrinciplesOf Biochemistry, at 793-800 (Worth Pub. 1982).

“Nucleic acid” refers to a polymeric form of nucleotides of any length,either ribonucleotides, deoxyribonucleotides or peptide nucleic acids(PNAs), that comprise purine and pyrimidine bases, or other natural,chemically or biochemically modified, non-natural, or derivatizednucleotide bases. The backbone of the polynucleotide can comprise sugarsand phosphate groups, as may typically be found in RNA or DNA, ormodified or substituted sugar or phosphate groups. A polynucleotide maycomprise modified nucleotides, such as methylated nucleotides andnucleotide analogs.

Indeed, the present invention contemplates any deoxyribonucleotide,ribonucleotide or peptide nucleic acid component, and any chemicalvariants thereof, such as methylated, hydroxymethylated or glucosylatedforms of these bases, and the like. The polymers or oligomers may beheterogeneous or homogeneous in composition, and may be isolated fromnaturally-occurring sources or may be artificially or syntheticallyproduced. In addition, the nucleic acids may be DNA or RNA, or a mixturethereof, and may exist permanently or transitionally in single-strandedor double-stranded form, including homoduplex, heteroduplex, and hybridstates.

The term “nucleic acid library” or sometimes “array” as used herein,refers to an intentionally created collection of nucleic acids which canbe prepared either synthetically or biosynthetically and screened forbiological activity in a variety of different formats (for example,libraries of soluble molecules; and libraries of oligos tethered toresin beads, silica chips, or other solid supports). Additionally, theterm “array” is meant to include those libraries of nucleic acids whichcan be prepared by spotting nucleic acids of essentially any length (forexample, from 1 to about 1000 nucleotide monomers in length) onto asubstrate.

The sequence of nucleotides may be interrupted by non-nucleotidecomponents. Thus the terms nucleoside, nucleotide, deoxynucleoside anddeoxynucleotide generally include analogs such as those describedherein. These analogs are those molecules having some structuralfeatures in common with a naturally occurring nucleoside or nucleotidesuch that when incorporated into a nucleic acid or oligonucleosidesequence, they allow hybridization with a naturally occurring nucleicacid sequence in solution. Typically, these analogs are derived fromnaturally occurring nucleosides and nucleotides by replacing and/ormodifying the base, the ribose or the phosphodiester moiety. The changescan be tailor made to stabilize or destabilize hybrid formation orenhance the specificity of hybridization with a complementary nucleicacid sequence as desired.

The term “hybridization” as used herein, refers to the process in whichtwo single-stranded polynucleotides bind non-covalently to form a stabledouble-stranded polynucleotide; triple-stranded hybridization is alsotheoretically possible. The resulting (usually) double-strandedpolynucleotide is a “hybrid.” The proportion of the population ofpolynucleotides that forms stable hybrids is referred to herein as the“degree of hybridization.”

Hybridizations are usually performed under stringent conditions, forexample, at a salt concentration of no more than 1 M and a temperatureof at least 25° C. For example, conditions of 5×SSPE (750 mM NaCl, 50 mMNaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30° C. aresuitable for allele-specific probe hybridizations. For stringentconditions, see, for example, Sambrook et al., Molecular Cloning Alaboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989), hereinincorporated by reference in its entirety.

The term “hybridization conditions” as used herein will typicallyinclude salt concentrations of less than about 1M, usually less thanabout 500 mM, and preferably less than about 200 mM. When the term“effective amount” is used herein, it refers to an amount sufficient toinduce a desired result. Hybridization temperatures can be as low as 5°C., but are typically >22° C., more typically >30° C., andpreferably >37° C. Longer sequence fragments may require higherhybridization temperatures for specific hybridization. Other factors mayaffect the stringency of hybridization, including base composition andlength of the complementary strands, presence of organic solvents andextent of base mismatching, as a result the combination of parameters ismore important than the absolute measure of any one alone.

The term “hybridization probe” as used herein, refers to anoligonucleotide capable of binding in a base-specific manner to acomplementary strand of nucleic acid. Such probes include peptidenucleic acids, as described in Nielsen et al., Science 254, 1497-1500(1991), and other nucleic acid analogs and nucleic acid mimetics. Theterm “hybridizing specifically to” as used herein, refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence or sequences under stringent conditions when thatsequence is present in a complex mixture (for example, total cellular)DNA or RNA.

The term “target” as used herein refers to a molecule that has anaffinity for a given probe. Targets may be naturally-occurring orman-made molecules. Also, they can be employed in their unaltered stateor as aggregates with other species. Targets may be attached, covalentlyor noncovalently, to a binding member, either directly or via a specificbinding substance. Examples of targets which can be employed by thisinvention include, but are not restricted to, antibodies, cell membranereceptors, monoclonal antibodies and antisera reactive with specificantigenic determinants (such as, on viruses, cells or other materials),drugs, oligonucleotides, nucleic acids, peptides, cofactors, lectins,sugars, polysaccharides, cells, cellular membranes, and organelles.Targets are sometimes referred to in the art as anti-probes. As the termtargets is used herein, no difference in meaning is intended. A “probeto-target pair” is formed when two macromolecules have combined throughmolecular recognition to form a complex (also referred to in the art areceptor-to-ligand binding).

The term “complementary” as used herein, refers to the hybridization orbase pairing between nucleotides or nucleic acids, such as, forinstance, between the two strands of a double stranded DNA molecule orbetween an oligonucleotide primer and a primer binding site on a singlestranded nucleic acid to be sequenced or amplified. Complementarynucleotides are, generally, A and T (or A and U), or C and G. Two singlestranded RNA or DNA molecules are said to be complementary when thenucleotides of one strand, optimally aligned and compared and withappropriate nucleotide insertions or deletions, pair with at least about80% of the nucleotides of the other strand, usually at least about 90%to 95%, and more preferably from about 98 to 100%.

Alternatively, complementarity exists when an RNA or DNA strand willhybridize under selective hybridization conditions to its complement.Typically, selective hybridization will occur when there is at leastabout 65% complementary over a stretch of at least 14 to 25 nucleotides,preferably at least about 75%, more preferably at least about 90%complementary. See, e.g., Kanehisa, Nucleic Acids Res. 12:203 (1984),incorporated herein by reference.

The microarray assay process is referred to herein as a Double-strandedExonuclease Protection (DEP) assay. The term “array” as used herein,refers to an intentionally created collection of molecules which can beprepared either synthetically or biosynthetically. The molecules in thearray can be identical or different from each other. The array canassume a variety of formats, such as for example, libraries of solublemolecules; libraries of compounds tethered to resin beads, silica chips,or other solid supports. The term “solid support,” “support,” and“substrate” as used herein, are used interchangeably and refer to amaterial or group of materials having a rigid or semi-rigid surface orsurfaces. In the exemplified embodiment the substrate is a glass slide.In many embodiments, at least one surface of the solid support will besubstantially flat, although in some embodiments it may be desirable tophysically separate synthesis regions for different compounds with, forexample, wells, raised regions, pins, etched trenches, or the like.According to other embodiments, the solid support(s) will take the formof beads, resins, gels, microspheres, or other geometric configurations.See, e.g., U.S. Pat. No. 5,744,305 for other exemplary substrates.

DEP employs DNA oligonucleotides (“oligomers”) for spotting onglass-slides to establish the microarrays. The DNA oligonucleotides(referred to simply as an “oligonucleotide” or “oligomers”) as usedherein, means a nucleic acid ranging from at least 2, preferable atleast 8, and more preferably at least 10-15, and more preferably atleast 20-30 nucleotides in length, or a compound that specificallyhybridizes to a polynucleotide. Polynucleotides of the present inventioninclude sequences of deoxyribonucleic acid (DNA) or ribonucleic acid(RNA) which may be isolated from natural sources, recombinantly producedor artificially synthesized and mimetics thereof. A further example of apolynucleotide of the present invention may be peptide nucleic acid(PNA). The invention also encompasses situations in which there is anontraditional base pairing, such as Hoogsteen base pairing, which hasbeen identified in certain tRNA molecules and are postulated to exist ina triple helix. “Polynucleotide” and “oligonucleotide” are usedinterchangeably in this application.

The oligonucleotides used in the present invention can be individuallyprepared by one of ordinary skill in the art, or they may be purchased,since many are commercially-available. In the present invention, thepreferred oligonucleotides include biotinylated thymidine residues.

The term “monomer” as used herein, refers to any member of the set ofmolecules that can be joined together to form an oligomer or polymer.The set of monomers useful in the present invention includes, but is notrestricted to, for example, (poly)peptide synthesis, the set of L-aminoacids, D-amino acids, or synthetic amino acids. As used herein,“monomer” refers to any member of a basis set for synthesis of anoligomer. For example, dimers of L-amino acids form a basis set of 400“monomers” for synthesis of polypeptides. Different basic sets ofmonomers may be used at successive steps in the synthesis of a polymer.The term “monomer” also refers to a chemical subunit that can becombined with a different chemical subunit to form a compound largerthan either subunit alone.

The term “isolated nucleic acid” as used herein, mean an object speciesinvention that is the predominant species present (i.e., on a molarbasis it is more abundant than any other individual species in thecomposition). Preferably, an isolated nucleic acid comprises at leastabout 50, 80 or 90% (on a molar basis) of all macromolecular speciespresent. Most preferably, the object species is purified to essentialhomogeneity (contaminant species cannot be detected in the compositionby conventional detection methods). The term “mixed population” or“complex population” as used herein, refers to any sample containingboth desired and undesired nucleic acids. As a non-limiting example, acomplex population of nucleic acids may be total genomic DNA, totalgenomic RNA or a combination thereof. Moreover, a complex population ofnucleic acids may have been enriched for a given population, but alsoinclude other undesirable populations. For example, a complex populationof nucleic acids may be a sample which has been enriched for desiredmessenger RNA (mRNA) sequences, but still includes some undesiredribosomal RNA sequences (rRNA). The oligonucleotide spots are preferablyisolated nucleic acids.

The term “primer” as used herein, refers to a single-strandedoligonucleotide capable of acting as a point of initiation fortemplate-directed DNA synthesis under suitable conditions for example,buffer and temperature, in the presence of four different nucleosidetriphosphates and an agent for polymerization, such as, for example, DNAor RNA polymerase or reverse transcriptase. The length of the primer, inany given case, depends on, for example, the intended use of the primer,and generally ranges from 15 to 30 nucleotides. Short primer moleculesgenerally require cooler temperatures to form sufficiently stable hybridcomplexes with the template. A primer need not reflect the exactsequence of the template but must be sufficiently complementary tohybridize with such template. The primer site is the area of thetemplate to which a primer hybridizes. The primer pair is a set ofprimers including a 5′ upstream primer that hybridizes with the 5′ endof the sequence to be amplified and a 3′ downstream primer thathybridizes with the complement of the 3′ end of the sequence to beamplified.

In practice, each spot on the microarray slide contains oneoligonucleotide arranged in the following orientation:

constant portion varying portion Slide - - - 5′ end-m13 - - - (spacer 20nucleotides) - - - biotinylated - - - antisense thymidine sequence-3′

The present microarray-based technique is effective on anysingle-stranded (ss) molecule, including single-stranded DNA or RNA, butcollectively is referred to herein as an “RNA sample,” specificallyincluding mRNAs. The term “mRNA” or sometimes refer by “mRNAtranscripts” as used herein, include, but not limited to pre-mRNAtranscript(s), transcript processing intermediates, mature mRNA(s) readyfor translation and transcripts of the gene or genes, or nucleic acidsderived from the mRNA transcript(s). Transcript processing may includesplicing, editing and degradation. As used herein, a nucleic acidderived from an mRNA transcript refers to a nucleic acid for whosesynthesis the mRNA transcript or a subsequence thereof has ultimatelyserved as a template. Thus, a cDNA reverse transcribed from an mRNA, anRNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNAtranscribed from the amplified DNA, etc, are all derived from the mRNAtranscript, and detection of such derived products is indicative of thepresence and/or abundance of the original transcript in a sample. Thus,mRNA derived samples include, but are not limited to, mRNA transcriptsof the gene or genes, cDNA reverse transcribed from the mRNA, cRNAtranscribed from the cDNA, DNA amplified from the genes, RNA transcribedfrom amplified DNA, and the like.

As the term RNA is used herein, it is further intended that the termalso encompasses other single-stranded nucleic acids, including ss-DNA.Likewise, although the process referred to is RNA expression profiling,it is further intended to include other single-stranded nucleic acids,including ss-DNA.

When the microarray assay is used for RNA expression profiling inaccordance with the present invention, the hybridization solution isapplied to the slide without prior amplification or labeling.Hybridization occurs in standard hybridization buffers and conditions.During incubation of the hybridization treated slide, the RNAhybridizing to the antisense portion (3′ portion) of the oligonucleotidewill produce a double-stranded DNA-RNA hybrid (or series of shortdouble-stranded DNA-RNA hybrids). By comparison, spots that containsingle-stranded antisense DNA oligonucleotides are not hybridized, thusthose oligonucleotides remain single-stranded.

Following hybridization, the slide is washed. Then, using a buffersolution that has been modified slightly from the commercialrecommendations, the slide is incubated overnight with Exonuclease I(New England Biolabs). Exonuclease I catalyzes the hydrolysis ofsingle-stranded DNA oligonucleotides to form free nucleotide residues.Hence for the (non-hybridized) single-strandedoligonucleotide-containing microarray spots, the biotinylated thymidineresidue will be cut away from the microarray, whereas the (hybridized)double-stranded oligonucleotide:RNA-containing microarray spots continueto include the biotinylated thymidine residue (i.e., the exonucleasedigests the single-stranded-DNA oligonucleotide).

Following incubation with Exonuclease I, the slides are again washed,and then incubated for 30 minutes at room temperature with a solutioncontaining streptavidin-conjugated Alexa fluor dye (SCAF) (MolecularProbes). The SCAF binds avidly to the biotinylated residues in themicroarray-bound oligonucleotides. As a result, SCAF only binds to thoseoligonucleotides that bound to the RNA sample (i.e., those that wereprotected from the single-stranded-DNA-specific Exonuclease I).Consequently, in the DEP assay, only the SCAF-bound oligonucleotidespots are fluorescent when the slide is evaluated by the detectiondevice, e.g., the slide scanner machine.

Of course, Exonuclease I is used only as an example of an effectiveexonuclease in the DEP assay. In fact, any exonuclease that effectivelycatalyzes hydrolysis of non-hybridized single-stranded oligonucleotidesin the spot to form free nucleotide residues, may be used provided thatthey (1) satisfy the basic criteria of strictly catalyzing3′→5′-directed nucleotide hydrolysis; (2) are independent of nucleotideidentity; and (3) work under conditions in which stable DNA/RNA hybridsare retained.

SCAF is also used only as an example of an effective marker. Otherstreptavidin-conjugated fluorophores or other luminescent labels mayalso be used, so long as the labeling method is combined with theessential step of using the double-stranded DNA/hybridized RNA as aprotection assay in the presence of an exonuclease. This preserves aresidue that can be labeled by the fluorophore or other luminescentlabel. In general, the term “label” as used herein refers to aluminescent label, a light scattering label or a radioactive label.Fluorescent labels include, inter alia, the commercially availablefluorescein phosphoramidites, such as, Fluoreprime (Pharmacia),Fluoredite (Millipore) and FAM (ABI). See also U.S. Pat. No. 6,287,778.

Furthermore, in an alternative embodiment, the present technique couldbe augmented by co-hybridization with a probe that recognizes the spacerportion of the oligonucleotide. Such a probe would employ a fluorescentor luminescent marker that is different from SCAF, i.e., red vs. green,to allow for a rigorous normalization of the amount of oligonucleotideDNA present in each spot. It also allows for good compensation forspot-to-spot heterogeneity.

The term “probe” as used herein refers to a surface-immobilized moleculethat can be recognized by a particular target. See, e.g., U.S. Pat. No.6,582,908 for an example of arrays having all possible combinations ofprobes with 10, 12, and more bases. Examples of probes that can beinvestigated by this invention include, but are not restricted to,agonists and antagonists for cell membrane receptors, toxins and venoms,viral epitopes, hormones (e.g., opioid peptides, steroids, etc.),hormone receptors, peptides, enzymes, enzyme substrates, cofactors,drugs, lectins, sugars, oligonucleotides, nucleic acids,oligosaccharides, proteins, and monoclonal antibodies.

As previously noted, the Northern blot is considered the standard methodfor miRNA validation and quantification (Ambros et al., 2003). TheNortherns are theoretically straightforward and their use has beenwell-established in biomedical research. Northern blots offer bothquantitative and qualitative information, and unlike a microarrayexperiment, a Northern blot confirms the length of the hybridizedtranscripts. The Northern blots are, however, laborious, making themless useful or well-suited for high-throughput expression profiling.

Accordingly, the present invention further provides a microarrayplatform that enables high-throughput gene expression analyses of smallRNAs. Termed the RNA-primed Array-based Klenow Enzyme (RAKE) assay, thisembodiment of the invention provides a new tool with high sensitivityand specificity for miRNA profiling.

By comparison, the present RAKE assay is quite simple, involving minimalsteps, and it is particularly suited for high-throughput expressionprofiling. The RAKE assay also provides unique qualitative data, becausethe 3′ end of the miRNA “primer” hybridizes specifically to theoligonucleotide “template.” As a result, RAKE appears to be superior toNorthern blots in discriminating the exact 3′ end of the sample miRNAs,offering a significant advantage because for many mature miRNAs, theparalogs differ at the 3′ end. These miRNAs, derived from differentgenes, would be expected to cross-react adversely in Northern blots (andin standard microarray methods using labeled target pools), but suchadverse reactions do not usually occur in the RAKE assay. As shown inFIGS. 7 and 8, data from hsa-miR-23a/23b (SEQID Nos: 1 and 2,respectively) and two other pairs of paralogous miRNAs, support thehypothesis that the RAKE assay is superior to Northern blots fordiscriminating miRNA paralogs.

Advantageously, RAKE requires no sample RNA manipulation. Possiblebiases, which may be introduced during enzymatic labeling, or cDNAgeneration or amplification of the sample RNA prior to hybridization tothe glass microarray, are thus avoided. Thus RAKE allows for rapid andsimultaneous detection of all known miRNAs from the same sample.Moreover, RAKE advantageously permits the complete automation of allsteps—from sample hybridization to detection. This is achieved by usinga number of existing technologies and equipment, previously used fortraditional mRNA microarrays, and allows for highly consistentperformance.

RAKE involves the generation of microarray-containing spottedoligonucleotides, wherein each DNA oligonucleotide is oriented on theslide essentially as set forth above for the DEP assay. The “constantportion” directly adjacent to the slide is the 5′ end of m13(spacer 20nucleotides) including thymidine residues, next to that is the “variableportion” comprising the antisense sequence-3′.

The RAKE technique involves a two-stage reaction. First, the microarrayis incubated along with Klenow enzyme, enzyme buffer (containing Mg⁺⁺),conjugated dATPs (for labeling), and an RNA sample. Some of the RNAsample hybridizes to the “antisense” (3′) sequence of the DNAoligonucleotide. Sequences that bind tightly, and are hybridized at the3′ end, can act as primers for the Klenow DNA polymerase (it has beendemonstrated that Klenow enzyme can act as a RNA-primer-directed DNApolymerase). In the present case, the Klenow fragment of DNA polymeraseI is applied to catalyze the addition of biotin-conjugated dNTPs, suchas dATPs, using the miRNA as a primer and the spotted probe as template.Since the incorporation of dideoxyNTPs (ddNTP) rather than dNTP is arandom event, the reaction will produce DNA fragments varying in length.In a preferred embodiment, the ratio of dNTP to ddNTP is selected togenerate DNA fragments of a predetermined size range. For example, DNAfragments sized may range from 20 to 50, 35-75, or 50 to 200 bases.Hence, the dATPs that can be labeled, only hybridize to oligonucleotide“spots” on the microarray that are antisense to RNAs present in thesample.

Technical ‘tweaks’ could greatly enhance/broaden the potentialapplications for RAKE. For example, longer RNAs including mRNAs (fortranscript splice variant profiling) may be assessed in the microarrayassay using a nuclease that cleaves non-hybridized RNA. The potentialapplications for these techniques are very broad, but include expressionanalyses of microRNAs (any species), siRNAs (experimentally introducedor endogenous), and other small regulatory RNAs. Other potential RNAapplications include profiling and quantification of viruses, as well asother RNA profiling.

Moreover, RAKE could be made a great deal more sensitive as needed, byutilizing one of various “sandwich” or signal amplification techniques(e.g., streptavidin HRP, biotin antibodies, chemiluminescence, etc).This is analogous to the discussion regarding the DEP assay above.

The present invention utilizes glass slide substrates for themicroarrays, e.g., CodeLink glass slides, with amine-modified DNAoligonucleotide probes spotted robotically, and specifically immobilizedvia attachment at the 5′ end (described in greater detail in the Examplethat follows). This technique provided results that on a technicallevel, proved to be consistent with findings in prior microarrayexperiments. For RNA targets analyzed using DNA oligonucleotideprobe-based microarrays, the probe-to-target cross-hybridization is seenonly if the degree of theoretical cross-hybridization is >80% for 50-70mer probes (Dai et al., Nucleic Acids Res. 30:e86 (2002)), and 90% ormore for an ˜20 mer probe, depending upon variables that includehybridization conditions, probe G/C composition, and the location of the‘mismatched’ nucleotide(s) (Ramakrishnan et al., Nucleic et al., Appl.Acids Res. 30:e30 (2002); El Fantroussi, Environ. Microbiol.69:2377-2382 (2003); Koizumi et al., Appl. Environ. Microbiol.68:3215-3225 (2002); Liu et al., Environ. Microbiol. 3:619-629 (2001);Dorris et al., BMC Biotechnol. 3:6 (2003)), CodeLink slide microarrayshave been shown to demonstrate excellent hybridization characteristics(Ramakrishnan et al., 2002; Dorris et al., 2003).

The RAKE assay was devised to exploit the known ability of the Klenowenzyme fragment to act as a DNA polymerase using an RNA primer on a DNAoligonucleotide template (Huang et al., Nucleic Acids Res. 24:4360-4361(1996); Huang et al., Anal. Biochem. 322:269-274 (2003)). Prior studieshave demonstrated on-slide enzymatic reactions and primer extension(see, e.g., Nikiforov et al., Nucleic Acids Res. 22:4167-4175 (1994);Head et al., Nucleic Acids Res. 25:5065-5071 (1997)). However, directdetection of RNA hybridization (using RNA-primed DNA polymerase) has notbeen reported on a microarray, nor have the special properties of theKlenow enzyme been used on such microarray studies.

It was also necessary to use Exonuclease I, a 3′→5′, single-stranded,DNA-specific exonuclease, which is highly processive (Brody et al., J.Biol. Chem. 261:7136-7143 (1986)). It is important to note that theactivities of both Klenow enzyme and exonuclease (Brody et al., 1986)are independent of the sequence of their substrates. Systematic bias is,therefore, not introduced, and the results produced by the presentinvention demonstrate sensitivity to the level of 10 picograms (pg) oftarget miRNA, which is comparable to Northern blots Lim et al., 2003. Incontrast, however, RNA ligases are prone to bias because enzyme kineticschange with substrate sequence (see Ohtsuka et al., 1977, supra;Romaniuk et al., 1982, supra), producing an inaccurate representation ofthe miRNAs present in a target pool labeled by RNA ligase methods.

In alternative embodiments of the present invention, even greatersensitivity may be obtained using ‘sandwich’-type amplification, or amore sensitive labeling technique (e.g., gold particles for ResonanceLight Scattering). Quantification of miRNA abundance, and resolution ofexpression differences, may be improved by incorporating a standardreference that hybridizes to the spacer sequence and is detected with asecond scanner channel.

The results provided by the present invention have proven to be broadlycompatible with those produced using Northern blots, e.g., Sempere etal., (2004, supra), although the present methods are far more efficientand effective. In each sampled tissue, only a minority of miRNAs areexpressed at detectable levels a given time in a given tissue.

Some miRNAs appear to be widely expressed, including miR-98, let-7paralogs, miR-16, miR-26a, and miR-100. MiR-124a and miR-9, which werefound only in samples from anaplastic oligodendrogliomas, are reportedlyhighly restricted, being expressed only in the central nervous system(Sempere et al., 2004, supra; Lim et al., 2003, supra). MiR-92, which ispresent in cultured cells, but not appreciably in the sampled tissues(Sempere et al., 2004), is also present in HeLa cells and Jurkat cells,but not in the tested primary tumor tissue described in the Example thatfollows. Finally, there is general agreement between the HeLa expressionprofile set forth herein as a result of the RAKE analysis, and themiRNAs evaluated previously in HeLa cells (Lagos-Quintana et al., 2001,supra; Mourelatos et al., Genes Dev. 16:720-728 (2002)). In the Examplethat follows, there was also the surprising finding that miR-20 showedstronger expression than one may have expected from prior studies. Thisexpression was validated by Northern blot (FIG. 7).

Jurkat cells are derived from a T cell lymphoma (Gillis et al., J. Exp.Med. 152:1709-1719 (1980)). In prior studies of chronic B celllymphomas, miR-15a and miR-16 were deleted or down-regulated in morethan two thirds of cases (Calin et al., 2002, supra), whereas miR-155was highly expressed in Burkitt lymphoma (Metzler et al., GenesChromosomes Cancer 39:167-169 (2004)). By contrast, Jurkat cells showstrong expression of both miR-15a and miR-16, and low expression ofmiR-155.

The sensitivity of the RAKE assay provides for sensitive, specific andhigh-throughput miRNA expression profiling, consistently produced robustsignals at 0.16 fmoles, equal to about 10⁸ molecules of miRNA. However,in contrast to the microarrays developed, e.g., by Liu et al., 2004,supra, and Miska et al., 2004, supra, the RAKE assay does not require orinvolve the generation of a cDNA library, nor is amplification of theRNA sample necessary. In fact, the RAKE assay avoids sample RNAmanipulation altogether. Moreover, the RAKE assay appears to be superiorto prior art methods for discriminating paralogous miRNAs that differ attheir 3′-ends, since the prior art techniques rely solely onhybridization to detect and discriminate between miRNA paralogs.

In addition, since blocks of formalin-fixed and paraffin-embedded (FFPE)tissue can be assessed using the RAKE technique (or other miRNA tools)as described in greater detail in the Example that follows, the presentinvention permits samples to be analyzed from the voluminous archive ofhuman pathological specimens. This will provide a better understandingof the roles of miRNAs in healthy and disease conditions.

The present invention can employ solid substrates, including arrays insome preferred embodiments. Methods and techniques applicable to polymer(including protein) array synthesis have been described in U.S. Ser. No.09/536,841, WO 00/58516, U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743,5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867,5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839,5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832,5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185,5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269,6,269,846 and 6,428,752, in PCT Applications Nos. PCT/US99/00730(International Publication No. WO 99/36760) and PCT/US01/04285(International Publication No. WO 01/58593), which are all incorporatedherein by reference.

The present invention also contemplates many uses for oligomers attachedto solid substrates. These uses include gene expression monitoring,profiling, library screening, genotyping and diagnostics. Geneexpression monitoring and profiling methods can be shown in U.S. Pat.Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248and 6,309,822. Genotyping and uses therefore are shown in U.S. Ser. Nos.10/442,021, 10/013,598 (U.S. Patent Application Publication20030036069), and U.S. Pat. Nos. 5,856,092, 6,300,063, 5,858,659,6,284,460, 6,361,947, 6,368,799 and 6,333,179. Other uses are embodiedin U.S. Pat. Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061, and6,197,506.

The practice of the present invention may also employ conventionalbiology methods, software and systems. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes and etc. The computer executable instructions may bewritten in a suitable computer language or combination of severallanguages. Basic computational biology methods are described in, forexample, Setubal et al., Introduction to Computational Biology Methods(PWS Publishing Company, Boston, 1997); Salzberg, Searles, Kasif, (Ed.);Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998);Rashidi and Buehler, Bioinformatics Basics: Application in BiologicalScience and Medicine (CRC Press, London, 2000) and Ouelette andBzevanis, Bioinformatics: A Practical Guide for Analysis of Gene andProteins (Wiley & Sons, Inc., 2^(nd) ed., 2001). See also, e.g., U.S.Pat. No. 6,420,108.

The present invention may also make use of various computer programproducts and software for a variety of purposes, such as probe design,management of data, analysis, and instrument operation. See, e.g., U.S.Pat. Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454,6,090,555, 6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.

Additionally, the present invention may have preferred embodiments thatinclude methods for providing genetic information over networks such asthe Internet as shown in U.S. Ser. Nos. 10/197,621, 10/063,559,10/065,856, 10/065,868, 10/328,818, 10/328,872, 10/423,403, and60/482,389.

Additional objects, advantages and novel features of the invention willbe set forth in part in the detailed protocols used as non-limitingexamples that follow, and in part will become apparent to those skilledin the art on examination of the following, or may be learned bypractice of the invention. The following examples, however, areunderstood to be illustrative only and are not to be construed aslimiting the scope of the appended claims.

EXAMPLES

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV); Using Antibodies: A Laboratory Manual; Cells: A LaboratoryManual; PCR Primer: A Laboratory Manual; and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, (1995) Biochemistry (4th Ed.) Freeman, N.Y.; Gait,“Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London; Nelson and Cox (2000); Lehninger, Principles of Biochemistry 3rdEd., W.H. Freeman Pub., New York, N.Y.; and Berg et al. (2002)Biochemistry, 5th Ed., W.H. Freeman Pub., New York, N.Y., all of whichare herein incorporated in their entirety by reference for all purposes.

Methods for conducting polynucleotide hybridization assays have beenwell developed in the art. Hybridization assay procedures and conditionswill vary depending on the application and are selected in accordancewith the general binding methods known including those referred to in:Maniatis et al., Molecular Cloning: A Laboratory Manual (2^(nd) Ed. ColdSpring Harbor, N.Y, 1989); Berger and Kimmel, Methods in Enzymology,Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc.,San Diego, Calif., 1987); Young and Davis, P.N.A.S, 80: 1194 (1983).Methods and apparatus for carrying out repeated and controlledhybridization reactions have also been described in U.S. Pat. Nos.5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of whichare incorporated herein by reference.

The present invention also contemplates signal detection ofhybridization between ligands in certain preferred embodiments. See,e.g., U.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758;5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639;6,218,803; and 6,225,625, in U.S. Ser. No. 10/389,194 and in PCTApplication PCT/US99/06097 (published as WO99/47964), each of which alsois hereby incorporated by reference.

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. Nos. 10/389,194,60/493,495 and in PCT Application PCT/US99/06097 (published asWO99/47964), each of which also is hereby incorporated by reference.

RNA Isolation and Northern Blots.

Cell batches (from HeLa and Jurkat cells) and RNA were processedseparately for biological replicates. RNA was purified initially usingTrizol™ LS reagent (InVitrogen, Carlsbad, Calif.). Human tissue wasprocured in accordance with accepted procedures. From archival FFPEtissue blocks, RNA was initially isolated essentially as previouslydescribed (Korbler et al., Exp. Mol. Pathol. 74:336-340 (2003)).Briefly, Citrisolv™ (Fisher Scientific, Pittsburgh, Pa.) was used as axylene substitute, and Trizol™ LS was used after deparaffinization.CitriSolv™ clearing/deparaffinizing agent, is nontoxic andbiodegradable, and provided results comparable with xylene.

Fresh tissues from two different brain tumors (a malignant meningiomaand an anaplastic oligodendroglioma) were dissected by aneuropathologist. Fresh tissue was placed in a RNALater™ solution(Ambion, Inc, Austin, Tex.). RNA was subsequently isolated from thisfresh tissue using Trizol™. For the anaplastic oligodendroglioma, theRNA from fresh tissue was compared against adjacent tissue from the sametumor that had been formalin-fixed and paraffin-embedded (FFPE) usingconventional anatomic pathology methods. For fresh tissue, culturedcells, and FFPE tissue, RNA was further processed using a kit designedto isolate low molecular weight RNA (MirVana™ kit from Ambion). ForNorthern blots, whole RNA was isolated from the cells or tissues usingthe Trizol™ LS reagent. RNA was then run on 20% urea-PAGE gels, blotted,and probed using 5′-end radiolabeled probes against the indicatedmiRNAs, as previously described by Nelson et al., RNA 10:387-394(2004)). Blots were exposed on phosphorimager screens overnight andsignals were scanned and quantified using a Storm 860 Phosphorimager(Molecular Dynamics, Sunnyvale, Calif.).

Microarray Platform.

Probe DNA oligonucleotides were synthesized at 600 pmol on 384-wellplates (Qiagen), each containing a 5′ terminal C6-amino modified linker.Each probe had a sequence as depicted in FIG. 1 (5′linker, m13-like‘spacer’, thymidines, and sequence antisense to miRNAs), except for acontrol probe, which contained the spacer only. Probes were suspended at40 μM in 150 mM sodium phosphate buffer (pH 8.5; 200U/ml print buffer)with 0.0005% Sarkosyl.

A GeneMachines OmniGrid 100™ robot printed probes onto CodeLink™ slides(Amersham Bioscience, Piscataway, N.J.) at 30-35% humidity at 24-27° C.Each spot element measured 120 μm in diameter with center-to-centerspacing of 400 μm. Each glass slide contained 6 spots (three spatiallyseparated pairs) corresponding to each probe, for a total 1422 spotsincluding controls. Further chemical ‘blocking’ of the spotted glassslide was found to be unnecessary.

RAKE Protocol.

Small RNA hybridizations were found to optimally include more than 2 μgtotal mass per slide. Less can be used, but the signal was weaker forless-abundant miRNAs. Spotted microarray slides were processed using anautomated hybridization apparatus (Tecan HS4800, Tecan Trading AG,Switzerland), which greatly facilitated sample processing and allowednear-identical handling of all microarray slides throughout.

The concentrated hybridization buffer is composed of 15% formamide,15×SSC. DNA oligonucleotide spike-ins corresponding to plant(Arabidopsis thaliana) miRNAs ath-miR-157, ath-miR-163, and ath-miR-169prepared beforehand in a solution at 10⁻⁷ M, 10−⁸ M, and 10⁻⁹ M,respectively, diluted in water. For all hybridization and enzymaticsteps, RNAsin (0.4U/μl; Promega Biosciences, Inc., San Luis Obispo,Calif.) was included. The protocol involves the following sequence: 1minute wash in 2×SSC at 25° C.; 5 minute rinse in 5×SSC with 10%formamide at 25° C.; 3×30 second rinses in 2×SSC at 25° C.; 18 hourtarget/probe hybridization (35 μl concentrated hybridization buffer, 65μl small RNA preparation containing 4 μg low molecular weight RNA, and10 μl plant DNA spike-in solution, which were together heated to 75° C.and allowed to cool at room temperature prior to hybridization) at 25°C. This was followed by 3×1 minute rinses in 2×SSC at 37° C.; 3 hourincubation with Exonuclease I (New England Biolabs, Ipswich, Mass.;fresh buffer at pH 7.5; 4U/pi) at 27° C.; 3×1 min. rinses in 2×SSC at27° C.; 10 minute rinse in 2×SSC with 0.05% SDS at 27° C.; 4×1 minuterinse in 2×SSC at 37° C.; 60 minute incubation with Exo(−) Klenow(Promega; 0.15 U/μl) in 1×DNA polymerase buffer (Promega) withbiotin-7-dATP (InVitrogen; 4 μM) at 27° C.; 2×1 min. rinse in 2×SSC at25° C.; 30 min incubation with streptavidin-conjugated Alexa-fluor-547(Molecular Probes; 15 ng/μl) at 25° C.; 3×1 min. rinses in 2×SSC at 25°C.

Validation Steps.

A concentration curve was generated using a synthetic target RNAoligonucleotide (miR-124a) in the background of a complex RNA mixture(low molecular weight RNA isolated from HeLa cells, a cell line thatdoes not contain miR124a). Plant miRNA spike-ins were used atconcentrations listed above.

For validating Northern blots, all of the miRNAs that were tested areset forth in FIG. 7.

Image Analysis and Data Processing.

Slides were scanned using a Genepix 4000B laser scanner (Axon, MolecularDevices, Sunnyvale, Calif.) at a constant power level and sensitivity(550 PMT) using a single color channel (532 nm wavelength).Non-hybridizing and artifact-associated spots were eliminated by bothvisual- and software-guided flags. Image intensities were measured as afunction of the median of foreground minus background. Negative valueswere normalized to zero, but no other normalizations were performed.Images were analyzed using the Genepix Pro5.0 software package. Exceland Genespring 6.2 were used for further data analysis. Testingdiscrimination of miRNA paralogs.

RNA oligonucleotides corresponding to 3 different miRNA paralogous pairs(6 different miRNAs; FIG. 8A) were synthesized and purified by PAGE.Corresponding antisense DNA oligonucleotides were also made, and servedas probes in Northern blots. RAKE experiments were performed using 0.1pmoles (˜6×10¹⁰ molecules) of each synthetic miRNA.

Hybridizations were performed in a complex mixture containing 2 μg ofsmall RNA from Jurkat cells (which do not normally express any of these6 synthetic miRNAs; see FIG. 5). Microarray experiments contained 6spots (probes) for each synthetic miRNA and were performed in duplicate(12 spots total for each synthetic miRNA). 0.1 pmoles of each syntheticmiRNA was fractionated on 15% urea-PAGE for Northern blots, which wereperformed in duplicate. Detection and signal quantification for RAKEassays and Northern blots were performed as described above.

Assay Development and Validation.

A method was developed to achieve high-throughput gene expressionanalyses of miRNAs. To eliminate systematic bias associated with RNAligation steps, amplification/cDNA intermediaries, or separatefluorophore labeling, on-slide enzymatic reactions, methods recognizedin the art for other purposes, were used.

The assay is shown in schematic form in FIG. 1. DNA oligonucleotideprobes, having 3′-halves complementary to specific miRNAs (e.g., let-7a)and having shared 5′-halves (spacer), were synthesized and covalentlycross-linked at their 5′ termini onto glass microarray slides. Threethymidines separated the spacer from the remainder of the DNA probe,which was antisense to specific miRNAs. The RNA sample, containingmiRNAs, was hybridized and after washes the slide was treated withExonuclease I, which specifically degraded single stranded,unhybridized, probes. The slide was again washed and the Klenow fragmentof the DNA polymerase I was applied along with biotinylated dATP(B-dATP). While any biotin-conjugated dATP may be used, and it isavailable in many places, in the present example, biotin-7-dATP(InVitrogen) was used. The hybridized miRNAs act as primers for theKlenow enzyme and the immobilized DNA probe acts as a template, leadingto incorporation of B-dATPs. The slide was then washed and astreptavidin-conjugated fluorophore was applied to visualize and analyzethe spots containing hybridized and Klenow-extended miRNAs.

In principle, either the Exonuclease I reaction (protection of a tagged,immobilized probe from nuclease by hybridization to a miRNA) or theKlenow DNA polymerase (primer extension from the hybridized miRNA on animmobilized probe template) should be effective alone to produceaccurate microarray-based detection of miRNAs. However, microarrays andprotocols designed to use either of these enzymes without the other,resulted in high background signal levels. It was subsequentlydetermined that the sequential application of these enzymes, asdescribed above (FIG. 1 and below) was optimal. A glass slide microarraywas developed, including probe spots corresponding to 239 miRNAs(sequences were obtained from the official microRNA registry(Griffiths-Jones, Nucleic Acids Res. 32 Database issue, D109-111 (2004))spotted in three pairs throughout the slide for a total of 1422 spotsper microarray, including miRNAs from humans, mice, rats, andArabidopsis thaliana.

Included also were DNA probes complementary to plant miRNAs on themicroarray, both for negative controls and for future studies involvingArabidopsis thaliana. Three separate Arabidopsis miRNA spike ins wereused. Synthetic DNA oligonucleotides corresponding to the three plantmiRNAs (ath-miR-157, ath-miR-163, and ath-miR-169) were introduced ineach hybridization step. For each hybridization, the final concentrationof ath-miR-157, ath-miR-163, and ath-miR-169 were 10⁻⁹ M, 10⁻¹⁰ M, and10⁻¹¹ M, respectively. These spike-in DNA oligonucleotides were used toassist normalization, and to provide absolute reference points for eachstudy (see below and FIG. 2). AthmiR-157, at 10⁻⁹ M (6.02×10¹⁰ probemolecules/100 μl hybridization reaction), provided an internal controlfor the highest fluorescent signal level.

The sensitivity of the RAKE assay was investigated by using a syntheticRNA target oligonucleotide corresponding to the sequence of maturemiR-124a. The results are shown in FIG. 2A, with each concentrationrepresenting duplicate arrays (12 data points each). Low molecularweight (LMW) RNA isolated from HeLa cells was included to compose acomplex RNA background. MiR-21 (a normal component of HeLa cells) andthe miR-157 DNA spike-in did not vary significantly at differentconcentrations of miR-124a (FIG. 2A). The dynamic signal of miR-124aspanned at least three orders of magnitude (FIG. 2A). These results werenot normalized, and thus demonstrate the robust nature of the raw data.

The technique is comparably sensitive to Northern blots (FIG. 2B),allowing detection of miRNA in the <10 pg range, which is consistentwith prior studies using Northern blots on miRNAs. The microarray datashows slightly less sensitivity, some variability at very lowconcentrations, and slightly less linearity across the large scale ofconcentrations when evaluated (five orders of magnitude), in comparisonto Northern blots (FIG. 2C). These differences are due to differentsaturation profiles inherent to microarray fluorescence, as comparedwith radiation detection.

RNA Isolation and Processing for RAKE.

RNAs derived from human epithelial and hematopoietic cell lines wereevaluated, as well as RNAs derived from two human brain tumors (Table1).

TABLE 1 Summary of tissues used in RNA studies. Amount of cellsCells/Tissue Designation Tissue Types or tissue needed HeLa Humanepithelial cancer cell line 10⁶ cells Jurkat Human T-cell derived 10⁶cells lymphoma cell line Malignant Meningioma Fresh human brain tumor<500 μg Anaplastic oligodendroglioma Fresh human brain tumor <500 μg(AO-Fresh) AO-FFPE Same tissues as above, formalin- 1-4. 50 μm thicksections fixed, paraffin embedded For HeLa cells, see Hsu, Tex. Rep.Biol. Med. 12: 833-846 (1954); Scherer et al., J. Exp. Med. 97: 695-710(1953). For Jurkat cells, see Gillis et al., J. Exp. Med. 152: 1709-1719(1980). For Anaplastic oligodendroglioma (AO-Fresh), tissue was adjacentto AO-P section.

In order to minimize the likelihood of cross-hybridization, only LMW RNAwas used in the microarray hybridizations. LMW RNA was isolated using acommercially available kit that successfully separated smaller fromlarger RNA species. The resulting LMW RNAs from the FFPE tissue appearedrelatively less degraded than the larger RNAs from FFPE tissue (FIG. 3).It was also considered that miRNAs could be isolated from archivalformalin-fixed, paraffin-embedded (FFPE) pathological material sinceshort segments of RNAs have been shown to be preserved in FFPE tissue(Van Deerlin et al., Neurochem. Res. 27:993-1003 (2002)), and siRNAs arerelatively slow to degrade in vivo (Chiu et al., Mol. Cell. 10:549-561(2002)).

To directly compare the performance of RAKE with RNA isolated from freshor FFPE material, tissue was obtained from a surgically removed humanbrain tumor (anaplastic oligodendroglioma). RNA was isolated from halfof the specimen and the other half of the specimen was submitted forroutine FFPE processing. RNA was then isolated from 50 μM-thick serialsections from the paraffin block. Consistent with prior studies, RNAprepared from fresh tissue appeared to be less degraded than that ofFFPE tissue (FIG. 3). It was also found that an appreciable amount oftotal RNA (up to 10 μgrams) could be isolated from a single 50 μM-thicksection of FFPE tissue.

RAKE Analysis on Tissue Cultured, FFPE, and Fresh Brain Tumor RNA.

Specimens were analyzed using three replicates for all RNA samples:biological replicates for HeLa and Jurkat cells, and technicalreplicates for human brain tumor tissue. Technical replicates wereperformed on the human brain tumors. A statistically definitive miRNAprofile for individual tumor types will result from further analyses ofmore human data. Valid conclusions about the RNA sampled in thereplicates is set forth in greater detail elsewhere herein.

Most negatively-hybridizing spots exhibited less of a signal, ascompared with background (FIG. 4). Signal was defined as the median offoreground spot fluorescence at 532 nm wavelength, minus background(defined by surrounding pixel intensity). Negative values werenormalized to zero. Otherwise, no normalization was used since there wasonly a single dye, the number of samples was only 239, and values wereconsistent across the microarray slides, as described elsewhere herein.A summary of the mean of the three replicates for all five samples (RNAfrom HeLa cells, Jurkat cells, malignant meningioma, and fresh andFFPE-derived anaplastic oligodendroglioma tissue) is presented in FIG.5. Results from duplicated miRNAs, ath-miRNAs, and other controlssuggest that only ath-miR-319 showed nonspecific signal (positive insome experiments without RNA targets), due to a technical problem withthis particular probe.

The DNA spike-in oligonucleotides produced high signal, as expected.Some miRNAs demonstrated high signal in multiple tissues (e.g., miR-15,miR-16, let-7f), whereas others were relatively restricted to certaintissue types (e.g., miR-27b in HeLa cells (SEQID No:4), miR148 in Jurkatcells, miR-199b in malignant meningioma, and miR-9 in the fresh and FFPEanaplastic oligodendroglioma). Biological and technical replicates werehighly correlated (with coefficients of correlation, R²>0.9).Representative examples are shown in FIG. 6. Note also that the freshand FFPE anaplastic oligodendroglioma showed results that were highlycorrelated (R2>0.9) with each other, showing that formalin fixation andparaffin embedding did not significantly skew the miRNA profile.

Northern Blot Validation.

Northern blots were used to evaluate selected data obtained by RAKE(FIG. 7). These experiments were performed on total RNA. Although theresults were for the most part the same between RAKE and the Northernblots, there was some discrepancy. The largest discrepancy was formiR-23b, which by Northern blots showed robust signal for both HeLa andmalignant meningioma derived RNA. RAKE showed no detectable amounts ofmiR-23b in all samples. These results suggest that miR-23b expressionwas indeed low or absent in these tissues. However, in Northern blots,the miR-23b DNA oligonucleotide probe might cross-hybridize with theparalogous miRNA, miR-23a (miR-23a: 5′-aucacauugccagggauuucc (SEQ IDNO:1); mir23b: 5′-aucacauugccagggauuaccac (SEQ ID NO:2). By contrast,the RAKE assay discriminated the difference between miR-23a and miR-23bbecause, like many paralogs, miR-23a and miR-23b differ at their 3′ end.Consequently, they could only prime Klenow extension when they werehybridized to the appropriate, specific probe.

Discrimination of Paralogous miRNA's that Differ at the 3′-end.

To further test the ability of RAKE to discriminate between paralogousmiRNAs, parallel microarray and Northern blot experiments were performedon three separate paralogous miRNA pairs (FIG. 8A, SEQID Nos:1, 2 and4-7) that differed primarily at their 3′-ends (most differences betweenmiRNA paralogs are found at the 3′-ends). Each paralogous miRNA (0.1pmoles; 6×10¹⁰ molecules) was analyzed by RAKE and on Northern blots(FIGS. 8B, 8C). The mean signal intensity (expressed in log units) foreach miRNA was determined in RAKE (n=1.2) and in Northern blots (n=2). Aratio between the paralogous miRNAs was calculated for each pair (shownin FIG. 8D). The results of this analysis showed that RAKE is superiorto Northern blots in discriminating miRNA paralogs.

For example, the power of RAKE to discriminate between hsa-miR-23a/23bparalogs in a sample containing hsa-miR-23a (SEQID No:1), was 9.6 times(=13.9/1.44) that of the Northern blots. Similarly, the power of RAKE todiscriminate between hsa-miR-23a/23b paralogs in a sample containinghsa-miR-23b (SEQID No:2), was 10.6 times (0.85/0.08) that of theNorthern blots. These values are somewhat different for each miRNAparalog tested in FIG. 8. It was likely due to at least two factors.First, the number, identity and position of the nucleotide differencesbetween the paralogous miRNAs, may have influenced their hybridizationproperties. Second, the use of B-dATP in the RAKE assay may have reducedits power to discriminate between certain miRNA paralogs. As describedabove (FIG. 1), B-dATP was used along with Klenow enzyme to extendhybridized miRNAs. When hsa-miR-200b (present in the sample RNA)hybridized to the mmu-miR-200b probe (which is the antisense DNAsequence of mmu-miR-200b (SEQID No:7)), the Klenow enzyme incorporated asingle B-dATP molecule on the hsa-miR-200b (SEQID No:6). This is becausethe mmu-miR-200b probe contains a thymidine corresponding to the adenine(underlined in FIG. 8A) found in the 3′-end of mmu-miR-200b, but notfound in hsa-miR-200b.

For the same reason, hsa-miR-23a (SEQID No:1) gave a weak signal on RAKEon the hsa-miR-23b (SEQID No:2) spot (FIGS. 8A, B). In contrast, up tothree B-dATP molecules may be incorporated when hsa-miR-200b hybridizesto its own probe (because there are three consecutive thymidines,corresponding to “Spacer sequences” (FIG. 1, SEQID No:3) after the3′-end of the hsa-miR-200b DNA probe SEQID No:6), leading to a strongersignal on RAKE. This also applies to hsa-miR-23a (SEQID No:1) whenhybridized to its own probe. Nevertheless, RAKE is still superior toNorthern blots in discriminating paralogous miRNAs differing at the3′-ends (FIG. 8D). The discriminating power of RAKE towards paralogousmiRNAs that differ by the presence of additional adenine(s) at their3′-ends (such as, hsa-miR-23b (SEQID No:2) versus hsa-miR23a (SEQIDNo:2), also may be improved by using B-dNTPs, other than dATP, andmodifying the sequences of the spacer accordingly.

Accordingly, the invention has established a microarray platform toenable high-throughput gene expression analyses of small RNAs. Inaddition, the RAKE assay may have applications besides miRNA geneexpression profiling, and the ability to apply the Klenow enzyme (withhigh sensitivity and specificity) as a RNA or DNA-primed polymerase on amicroarray slide will open the door to further interesting studiesincluding, for example, viral gene expression profiling.

Each and every patent, patent application and publication that is citedin the foregoing specification is herein incorporated by reference inits entirety.

While the foregoing specification has been described with regard tocertain preferred embodiments, and many details have been set forth forthe purpose of illustration, it will be apparent to those skilled in theart without departing from the spirit and scope of the invention, thatthe invention may be subject to various modifications and additionalembodiments, and that certain of the details described herein can bevaried considerably without departing from the basic principles of theinvention. Such modifications and additional embodiments are alsointended to fall within the scope of the appended claims.

What is claimed is:
 1. An assay method for analyzing a nucleic acidsample, the assay method comprising; obtaining a single-stranded nucleicacid sample comprising at least one target RNA having an unknownexpression profile; immobilizing onto a substrate at least one knownsingle-stranded oligonucleotide hybridization probe, to provide at leastone immobilized probe template, the probe comprising a nucleotidesequence complementary to the at least one target RNA in thesingle-stranded nucleic acid sample, wherein the nucleotide sequence isfollowed 5′ by consecutive thymidine nucleotides; hybridizing the atleast one target RNA to the complementary nucleotide sequence of the atleast one immobilized probe template under hybridization conditions, toprovide at least one hybridized target RNA, thereby producing at leastone double-stranded substrate-immobilized hybridized nucleic acidcomprising the at least one hybridized target RNA and the at least oneimmobilized probe template, wherein the at least one hybridized targetRNA is a at least one hybridized target RNA primer and the at least oneimmobilized probe template is a at least one template for templatedirected synthesis; then washing the substrate containing thesubstrate-immobilized hybridized nucleic acid; incubating the at leastone substrate-immobilized hybridized nucleic acid with polymerase tocatalyze the addition of at least one labeled complementary nucleotideonto the 3′ end of the at least one hybridized target RNA primer,producing a primed extension product comprising at least oneincorporated nucleotide, wherein the at least one incorporatednucleotide is the at least one labeled complementary nucleotide; thenwashing to remove non-hybridized and unincorporated nucleotides;incubating the washed, primed extension product with a fluorescent,luminescent or radioactive detection reagent that binds to the at leastone labeled complementary nucleotide on the primed extension product;imaging the resulting extension product and signals from the detectionreagent bound thereto; and identifying image intensities to determine ifa recognition image intensity is present, indicating hybridizationbetween the at least one target RNA and the at least one immobilizedprobe template.
 2. The method of claim 1, further comprising quantifyingabundance of the at least one target RNA in the sample targeted by theat least one immobilized probe.
 3. The method according to claim 2,wherein the single-stranded nucleic acid sample comprises at least onemicroRNA.
 4. The method according to claim 1, wherein the at least oneincorporated nucleotide comprises a biotin label.
 5. The methodaccording to claim 2, wherein the at least one double-strandedsubstrate-immobilized hybridized nucleic acid is RNA/DNA.
 6. The methodaccording to claim 1, further comprising adding an exonuclease tohydrolyze a single-stranded probe template which does not hybridize tothe at least one target RNA.
 7. The method according to claim 6, whereinthe exonuclease is Exonuclease I.
 8. The method according to claim 1,wherein the polymerase is Klenow DNA polymerase.
 9. The method accordingto claim 1, wherein the luminescent label used to label the hybridizednucleic acids is a fluorophore.
 10. The method according to claim 9,wherein the fluorophore is a streptavidin-conjugated fluorophore. 11.The method of claim 1, wherein the substrate is a glass micro arrayslide.
 12. The method claim 1, providing high-throughput expressionprofiling.
 13. The method of claim 3, further comprising expressionprofiling the at least one microRNA based upon the resultinghybridization pattern.
 14. The method of claim 3, further comprisingviral gene expression profiling based upon the resulting hybridizationpattern.
 15. The method of claim 1, wherein absence of recognition imageintensity indicates absence of hybridization between the at least oneimmobilized probe template and the at least one target RNA.
 16. Themethod of claim 1, wherein depending upon the hybridization templatesthat are immobilized, the assay permits simultaneously detectingmultiple target RNAs in the sample.
 17. The method of claim 1, whereinthe assay is an RNA-primed Array-based Klenow Enzyme (RAKE) assay. 18.The method of claim 17, providing high-throughput expression profiling.19. The method of claim 17, providing viral gene expression profiling.